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The Three Essential Motifs in P0 for Suppression of RNA Silencing Activity of Potato leafroll virus Are Required for Virus Systemic Infection.

ABSTRACT: Higher plants exploit posttranscriptional gene silencing as a defense mechanism against virus infection by the RNA degradation system. Plant RNA viruses suppress posttranscriptional gene silencing using their encoded proteins. Three important motifs (F-box-like motif, G139/W140/G141-like motif, and C-terminal conserved region) in P0 of Potato leafroll virus (PLRV) were reported to be essential for suppression of RNA silencing activity. In this study, Agrobacterium-mediated transient experiments were carried out to screen the available amino acid substitutions in the F-box-like motif and G139/W140/G141-like motif that abolished the RNA silencing suppression activity of P0, without disturbing the P1 amino acid sequence. Subsequently, four P0 defective mutants derived from a full-length cDNA clone of PLRV (L76F and W87R substitutions in the F-box-like motif, G139RRR substitution in the G139/W140/G141-like motif, and F220R substitution in the C-terminal conserved region) were successfully generated by reverse PCR and used to investigate the impact of these substitutions on PLRV infectivity. The RT-PCR and western blot analysis revealed that these defective mutants affected virus accumulation in inoculated leaves and systemic movement in Nicotiana benthamiana as well as in its natural hosts, potato and black nightshade. These results further demonstrate that the RNA silencing suppressor of PLRV is required for PLRV accumulation and systemic infection.

SUBMITTER: Rashid MO

PROVIDER: S-EPMC6410027 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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The Three Essential Motifs in P0 for Suppression of RNA Silencing Activity of Potato leafroll virus Are Required for Virus Systemic Infection.

Rashid Mamun-Or MO Zhang Xiao-Yan XY Wang Ying Y Li Da-Wei DW Yu Jia-Lin JL Han Cheng-Gui CG

Viruses 20190220 2

Higher plants exploit posttranscriptional gene silencing as a defense mechanism against virus infection by the RNA degradation system. Plant RNA viruses suppress posttranscriptional gene silencing using their encoded proteins. Three important motifs (F-box-like motif, G139/W140/G141-like motif, and C-terminal conserved region) in P0 of Potato leafroll virus (PLRV) were reported to be essential for suppression of RNA silencing activity. In this study, Agrobacterium-mediated transien ...[more]

PMID: 30791535

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Project description:BackgroundCertification of seed potato as free of viruses is essential for stable potato production. Among more than 30 virus species infecting potato, potato leafroll virus (PLRV), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY) predominate worldwide and should be the targets of a high-throughput detection protocol for seed potato quarantine.ResultsWe developed an assay based on one-step real-time multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with melt curve analysis for the four viruses and one internal control, potato elongation factor 1 alpha gene (EF1α). Virus-specific primers were derived from conserved regions among randomly selected representatives considering viral genomic diversity. Our assay simultaneously detected representative Japanese isolates of PLRV, O lineage of PVS, PVX, and NTN strain of PVY. The variability of melting temperature (Tm) values for each virus was confirmed using Japanese isolates, and virus species could be identified by the values of 87.6 for PLRV, 85.9 for PVX, 82.2 (Ordinary lineage) to 83.1 (Andean lineage) for PVS, and 79.4 (NA-N strain) to 80.5 (O strain and NTN strain) for PVY on average. The reliability of calculation was validated by comparing the calculated Tm values and measured Tm values and the values had a strong linear correlation (correlation of determination: R2 = 0.9875). Based on the calculated Tm values, representative non-Japanese isolates could also be identified by our assay. For removing false positives, two criteria were set for the evaluation of result; successful amplification was considered as 30.0 ≥ threshold cycle value, and the virus-specific peak higher than the EF1α-specific peak was considered as positive. According to these criteria, our assay could detect PLRV and PVS from 100-fold dilution of potato leaf homogenate and PVX and PVY from 1000-fold in a model assay.ConclusionThis new high-throughput detection protocol using one-step real-time mRT-PCR was sensitive enough to detect viruses in a 100-fold dilution of singly-virus contaminated homogenate in a model assay. This protocol can detect the four viruses in one assay and yield faster results for a vast number of samples, and greatly save the labor for seed potato quarantine and field surveys.

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The Three Essential Motifs in P0 for Suppression of RNA Silencing Activity of Potato leafroll virus Are Required for Virus Systemic Infection.

Publications

The Three Essential Motifs in P0 for Suppression of RNA Silencing Activity of <i>Potato leafroll virus</i> Are Required for Virus Systemic Infection.

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